Alat rapid antigen: basic knowledge of nucleic acid detection - part 1

Alat rapid antigen: basic knowledge of nucleic acid detection

1. What is nucleic acid detection

Definition of nucleic acid: nucleic acid is a kind of biological macromolecules connected by nucleotides or deoxynucleotides through 3 ', 5' - phosphate diester bond.

Nucleic acid has very important biological functions, mainly storing and transmitting genetic information.

2. Classification of nucleic acids

Nucleic acid macromolecules can be divided into two categories: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

3. Composition of nucleic acid

DNA and RNA are formed by connecting the head and tail of a nucleotide, which is composed of five elements: C, h, O, N and P.DNA is the genetic material of most organisms. RNA is a genetic material of a few DNA free viruses, such as HIV, influenza, SARS virus, etc.The average length of RNA is about 2000 nucleotides, while human DNA is very long, about 3x10 ^ 9 nucleotides.

4. Function of nucleic acid

It plays a role in storing and transmitting genetic information in protein replication and synthesis.Nucleic acid is not only the basic genetic material, but also plays an important role in protein biosynthesis. Therefore, it plays a decisive role in a series of important life phenomena such as growth, heredity and variation.

DNA and RNA are nucleic acids. What are the differences in their chemical composition as follows:

5. Test method

Nucleic acid detection method mainly detects the target nucleic acid in the sample by simultaneously amplifying the target nucleic acid and generating the detectable signal.It can be applied to nucleic acid detection in clinical microbiology, blood screening, genetic disease diagnosis and prevention, forensic medicine and other fields.

At present, the main methods used are as follows:

a. Nucleic acid sequence dependent amplification

NASBA is a new technique for isothermal amplification of RNA mediated by a pair of primers, which is continuous and uniform and specific nucleotide sequence in vitro.The reaction was carried out at 42 ℃, and the RNA template could be amplified about 109 times in 2 hours.The principle of NASBA is to extract viral RNA, add AMV reverse transcriptase, RNase H, t7rna polymerase and primers for amplification.The whole reaction is divided into non cyclic phase and cyclic phase: in the non cyclic phase, primer I and template RNA are annealed to synthesize cDNA under the action of AMV reverse transcriptase to form RNA: DNA hybrid, then RNaseH degrades RNA, primer II and cDNA are annealed to synthesize the second DNA complementary strand under the action of reverse transcriptase.Under the action of t7rna polymerase, double stranded DNA can be started by its promoter sequence to transcribe RNA. RNA can be reverse transcribed into DNA under the action of reverse transcriptase, enter the circulating phase and amplify a large number of templates.

b. Transcription mediated amplification

The technical principle of TMA is basically the same as that of NASBA. The slight difference is that TMA uses MMLV reverse transcriptase and t7rna polymerase. MMLV reverse transcriptase has both reverse transcriptase activity and RNase H activity.The reaction was carried out at 41.5 ℃, and the RNA template could be amplified about 109 times in 1 hour.

c. Ligase catalyzed chain reaction (LCR)

LCR is a probe amplification technology based on the interconnection of target molecule dependent oligonucleotide probes. It is a promising in vitro amplification technology developed after PCR.Its principle is to hybridize the two segment oligonucleotide single stranded DNA probe with the target sequence. When the two segment DNA probe fades with the template without mutation, if the two probes are adjacent and there is no nucleotide interval in the middle, they can be connected under the action of ligase, and the new chain after connection can be used as a template to guide the connection in the next cycle to produce new sub chains.If the base mutation of nucleotide occurs in the connecting section, the connecting reaction cannot occur and the amplification reaction is terminated.

d. Polymerase chain reaction assay (PCR)

The basic principle of PCR technology is similar to the natural replication process of DNA, and its specificity depends on oligonucleotide primers complementary to both ends of the target sequence.PCR consists of three basic reaction steps: denaturation annealing extension.

① Denaturation of template DNA: after the template DNA is heated to about 93 ℃ for a certain time, the double strand of template DNA or the double strand DNA formed by PCR amplification is dissociated to form a single strand, so that it can be combined with primers to prepare for the next round of reaction.

② Annealing (refolding) of template DNA and primer: after the template DNA is heated and denatured into a single strand, the temperature drops to about 55 ℃, and the primer is paired with the complementary sequence of the single strand of template DNA.

③ Primer extension: under the action of taqdna polymerase, DNA template primer conjugate uses dNTP as reaction raw material and target sequence as template to synthesize a new semi retained replication chain complementary to the template DNA chain according to the principle of base pairing and semi retained replication. Repeat the three processes of cyclic denaturation annealing extension to obtain more "semi retained replication chains",And this new chain can become the template for the next cycle.It takes 2-4 minutes and 2-3 hours to amplify the target gene to be expanded millions of times.To detect HIV RNA, we need to transcribe RNA into cDNA by reverse transcription reaction, and then PCR amplification with cDNA as template. This reaction is called RT-PCR.